Mitsugumin 29 (or MG29) is a synaptophysin-family member protein localized specifically in the triad junction of skeletal muscle, where the transverse tubular (T-tubule) invagination of plasma membrane touches the terminal cistemae of sarcoplasmic reticulum (SR). The triad junction provides the structural framework for interaction between the dihydropyridine receptor/voltage sensor and the ryanodine receptor (RyR)/Ca release channel, and is essential for the operation of excitation-contraction (E-C) coupling. MG29 appears to be indispensable for triad formation, since swollen T-tubules and vacuolated SR networks are observed in skeletal muscle isolated from the mg29(-/-) mice. The long term goal of this project is to understand the cellular and molecular function of MG29 in E-C coupling of muscle cells, specifically to test the hypothesis that MG29 is a key protein involved in the biogenesis of the T-tubule system, and the proper organization of the T-tubule structure is essential for both short-term E-C coupling and long term cellular Ca homeostasis. A distinct phenotype of mg29(-/-) mice is their increased susceptibility to muscle fatigue, which may be linked to an altered Ca signaling in the mutant muscle. Aim 1 of this project is to correlate the MG29-mediated changes in membrane structure with short-term changes in voltage-induced Ca release (VICR) and Ca-induced Ca release (CICR) in skeletal muscle. The defective membrane structure associated with deletion of MG29 could alter the retrograde interaction between RyR and store-operated Ca channel (SOC) in the plasma membrane, and affect long-term Ca homeostasis in muscle cells. Aim 2 of this project will investigate the mechanism of SOC regulation in skeletal muscle by MG29, by exploring the potential interaction of MG29 with RyR, caveolin-3 and other associated proteins. Aim 3 is designed to further test the role of T-tubule structure in the differential mechanism of Ca signaling in cardiac and skeletal muscles. A transgenic mouse has been generated that switched the mg29 gene from skeletal to cardiac muscle (mhc. mg29). The changes in efficacy of E-C coupling in cardiac myocytes and skeletal myotubes isolated from the mhc-mg29 and mg29(-/-) mice will be compared with the wild type controls, in order to understand the contribution of T-tubules to Ca signaling in heart and skeletal muscle and the molecular basis of muscle fatigue. Overall, fulfillment of experiments proposed in this project shall lead us to better understanding of the molecular mechanism of E-C coupling, in particular with respect to the roles of MG29 and RyR in the activation of CICR, VICR, and SOC in cardiac and skeletal muscles. In addition, our studies with the mutant mice will provide new insights into the cellular mechanism of muscle exercise physiology and cardiovascular diseases.